Integrated aerobic exercise with LDE-docetaxel treatment: a novel approach to combat prostate cancer progression

The variability in response to conventional prostate cancer (PC) therapies, coupled with the emergent issue of drug resistance, underscores the critical need for innovative treatment strategies. Aerobic physical exercise reduced incidence of several cancers, but the mechanism underlying these effects associated the nanoemulsion not fully understood. The application of a lipid nanoemulsion (LDE) delivery system for docetaxel (DTX), showing marked enhancement in therapeutic efficacy when combined with aerobic physical exercise. This novel intervention potentiates the antitumor activity of LDE-delivered DTX by augmenting nanoparticle internalization and inducing cell cycle arrest. Our findings reveal that this synergistic treatment not only significantly reduces prostate weight and mitigates adenocarcinoma proliferation but also attenuates anti-apoptotic BCL-2 protein expression. Concurrently, it elevates pro-apoptotic proteins and diminishes inflammatory markers. Metabolic profiling of the combined therapy group disclosed additional benefits, such as reduced lipid and plasma glucose levels. Collectively, our data illuminate the profound impact of integrating LDE-mediated DTX delivery with structured physical exercise, which together spearhead a dual-front assault on PC. This multimodal approach heralds a new paradigm in PC management, accentuating the promise of combined pharmacological and non-pharmacological interventions to elevate tumor suppressor protein activity and refine patient outcomes.

mimicry and apo E binding, these nanoemulsions have shown substantial promise.When paired with taxanes like paclitaxel and docetaxel, LDEs significantly mitigate toxicity and enhance pharmacological efficacy 3 .
Clinical trials involving LDE-paclitaxel have yielded encouraging results in terminal-stage patients suffering from ovarian, breast, lung, and prostate cancers with bone metastasis, demonstrating negligible toxicity and suggestive therapeutic advantages.Additionally, docetaxel loaded LDEs have been explored in rabbit models of atherosclerosis induced by cholesterol diets, where they not only attenuated atherosclerotic lesions but also exhibited pronounced anti-inflammatory effects associated to LDE was tested only in rabbits with atherosclerosis induced by cholesterol feeding 5 .This innovative approach not only paves the way for more effective chemotherapeutic regimens but also underscores the potential of LDE-docetaxel in the broader context of inflammatory pathologies.
Physical exercise is well-documented to confer a multitude of benefits across various physiological domains, including metabolic regulation, cardiovascular health, and cellular plasticity [6][7][8] .Within the realm of oncology, a pivotal mechanism underlying the beneficial effects of exercise is its role in apoptotic modulation via the p53 pathway in prostate cells, suggesting a potential oncogenic attenuation [9][10][11] .Concurrently, exercise-induced lipid modulation has been implicated in the regulation of serum cholesterol and glucose concentrations, thereby potentially curtailing the metabolic support of neoplastic cells 12,13 .
Our previous research has established that aerobic physical exercise can attenuate the progression of prostate cancer 11,14 .The present inquiry seeks to elucidate the synergistic effects of physical activity when coupled with conventional prostate cancer treatments.In the context of lipid metabolism, it has been demonstrated that athletes exhibit a fivefold increase in the systemic clearance of LDE compared to sedentary individuals 15 .Further corroborating this, experimental models utilizing LDL receptor knockout mice subjected to exercise regimens have revealed an overexpression of LDL receptors, which may elucidate the enhanced clearance of LDE 16 .These findings advocate for a deeper investigation into the adjunctive role of physical exercise in the therapeutic management of prostate cancer.
We investigated the anti-cancer effectiveness of LDE-docetaxel, a lipid nanoemulsion that may enhance tumor targeting via LDL receptors and modulate apoptotic proteins.Our study delved into the interaction between nanoparticles and cancer cells, examining impacts on cell cycle and apoptosis, as well as tumor biomarkers in the blood.We considered the possibility that physical exercise might increase the uptake of LDE-docetaxel by cancer cells, thereby boosting its anti-cancer activity.Using a prostate cancer model, we found that LDE-docetaxel regulates oncogenic protein levels.Our data indicate that physical exercise amplifies the effects of LDE-docetaxel, leading to a reduction in prostatic adenocarcinoma.This study primarily aims to assess the anti-cancer potential of LDE-docetaxel in an animal model of prostate cancer and determine whether physical exercise can enhance its efficacy.Our findings suggest that combining physical training with LDL receptor-targeted chemotherapy may represent a novel and effective approach to prostate cancer treatment.

Body weight, feeding efficiency, tumoral volume and incremental load test
To study the effects of treatments on nutrition and body composition we analyzed weekly food consumption and weight of animals from the beginning to the end of the protocol.The body weight of the animals at the beginning was similar among the six groups (Fig. 1A).Throughout the intervention period, in the fourth, fifth and seventh week, the animals in the PC + Ex group had lower body weight when compared to the PC group (Fig. 1B).However, after experimental treatment period the final body weight was significantly lower in the PC + LDE-DTX group (95% CI 30.89-132.3; p = 0.0007), e PC + LDE-DTX + Ex (95% CI 19.54-124.1;p = 0.0041) when compared to the PC group (Fig. 1C).Weight gain over the experimental treatment period varied with a significant difference at second week between the PC groups and the PC + Ex group (95% CI 2.689-43.92;p = 0.0360), in the fourth week between the PC + Ex groups (95% CI 7.798-14.88;p = 0.0018), LDE-DTX (95% CI 5.368-8.226;p = 0.0006) and PC + LDE-DTX + Ex (95% CI 5.585-13.85;p = 0.0045) when compared respectively with the PC group (Fig. 1D).Final weight gain was lower in the PC + LDE-DTX group compared to the PC group (95% CI 7.060-112.6;p = 0.0218; Fig. 1E).
To identify the relationship between weight loss and the growth of the tumor mass, we verified the energy intake and calculated the feed efficiency, as well as analyzed the volume of the tumor in the face of the treatments.Energy intake in the PC + Ex group had a significant reduction over in the seventh week compared to PC (p = 0.0354) and PC + LDE-DTX groups (p = 0.0043; Fig. 1F).The PC + Ex group showed energy intake compared to the PC group (95% CI 4.904-33.43;p = 0.0053) and PC + LDE-DTX group (95% CI − 28.72 to − 0.1936; p = 0.0461; Fig. 1G).Feed efficiency did not differ between groups (Fig. 1H,I).Water consumption did not differ between groups (Fig. 1J).Body fat reduced in PC + Ex (95% CI 3.484-10.17;p < 0.0001), PC + LDE-DTX (95% CI 2.353-8.750;p = 0.0004) and PC + LDE-DTX + Ex (95% CI 3.213-10.29;p = 0.0001) compared to PC group (Fig. 1K).
During the induction period and experimental protocol, there was a loss of approximately 12% of the total sample.Animals treated with PC + LDE-DTX and PC + LDE-DTX + Ex groups showed an effective reduction in tumor volume in experimental treatment period compared to the PC group (1.96-folds to PC + Ex, 2.79-folds to PC + LDE-DTX, and PC + LDE-DTX + Ex reduced about 4.04-folds, respectively; Fig. 1L).Most notably, the absolute prostate weight was lower in the PC + Ex, PC + LDE-DTX, and PC + LDE-DTX + Ex groups (78%, 116%, and 123% respectively) compared to the PC (Fig. 1M).
We performed incremental tests in 3 moments during the experimental treatment period.The incremental load test was similar between groups at baseline moment.Between moments 1 and 2, the animals PC + Ex and PC + LDE-DTX + Ex groups showed better performance (Fig. 1N).When analyzing moment 3, there was a reduction in physical capacity in both groups, where the PC + Ex group presented lower physical capacity when Vol:.( 1234567890 1N,O).Therefore, aerobic physical exercise seems to have been more effective in maintaining physical capacity when associated with PC + LDE-DTX (Fig. 1N).

Raman spectroscopy and biochemistry
Figure 2 shows the Raman spectra of blood serum.To evaluate spectral variance across the groups, we used principal IDMAP, as shown in Fig. 2B.Our results illustrate good clustering for different treatments (Fig. 2A) with greater variation in Raman spectra.The computational projection technique groups by similarities, but not only considers the difference between the Raman spectra but also the shape of the spectra shown in Fig. 2A.All the assignments of the bands in low and high wavenumber are present in the Raman spectra.The intensity of the band 417 cm −1 cholesterol was lower in the PC + Ex groups (95% CI 1279-2255; p < 0.0001; Fig. 2A), PC + LDE-DTX (95% CI 792.2-1768; p < 0.0001; Fig. 2C) and PC + LDE-DTX + EX (95% CI 660.5-1636; p < 0.0001; Fig. 2C) compared to PC group (Fig. 2C).The sign of the intensity of the band 505 cm −1 that indicates the amount of glucose in blood was lower for the PC + LDE-DTX + Ex group when compared to the PC group (95% CI 7279-11,609; p < 0.0001; Fig. 2D) and PC + Ex group (95% CI 300.0-4631; p = 0.0244; Fig. 2D).Also, the PC group showed higher intensity values of the 505 cm −1 band when compared to the PC + Ex groups (95% CI 4813-9144; p < 0.0001; Fig. 2B) and PC + LDE-DTX (95% CI 5148-9479; p < 0.0001; Fig. 2D).

LDE uptake
Table 1 shows LDE uptake.It was possible to verify that the animals submitted to physical exercise presented greater uptake of the LDE nanoemulsion compared to the group without physical exercise, with significant differences between the liver (p = 0.001) and prostate (p = 0.03).

Histopathology analysis
To understand the effect of the nanoemulsion LDE conjugated to DTX associated or not with physical exercise in prostate cancer, we verified the histopathology of the prostate of rats submitted to LDE-docetaxel treatment and to aerobic physical exercise.Table 2 showed histopathology analysis.The PC group had 58.71% of highgrade intraepithelial neoplasia (HGPIN) and a lower amount of low-grade intraepithelial neoplasia (LGPIN,) when compared to the other groups (Table 2).The PC + LDE + DTX and PC + LDE-DTX + Ex groups showed a reduction in the amount number of acinus with HGPIN and greater expression of LGPIN, reducing the degree of severity of the development of neoplasms (Table 2).There were no differences between groups in the amount number of acinus with metaplasia.The PC group had an average of 10% of prostatic acini with adenocarcinoma.
Treatment with LDE-DTX in the PC + LDE + DTX and PC + LDE-DTX + Ex groups significantly reduced the number of acini with adenocarcinoma (Table 2).
To evaluate prostatic volume with LDE-DTX treatment and physical exercise, we used the Weibel method.Figure 3A,E shows LDE-docetaxel treatment reduced the volume of the epithelium compared to PC group (p = 0.0232).In addition, the PC group showed a lower connective tissue percentage compared to PC + Ex, PC + LDE-DTX, and PC + LDE + DTX + EX (Fig. 3A,F).The lumen percentage does not show significant differences (Fig. 3A,G).

Cell proliferation, cell cycle and apoptosis analysis
To investigate the direct effects of LDE-DTX in combination with physical exercise on cell proliferation, apoptosis, and cell cycle arrest, we employed immunofluorescence and immunohistochemistry staining techniques.Additionally, we quantified the expression levels of relevant proteins involved in these processes using Western blot analysis.

LDE + DTX increased the expression of Antioxidants
The mRNA levels of Nos2, Sod1, Cat, and Gss were analyzed in the prostate (Fig. 4).In the PC + LDE + DTX group, there was an increase in

Inflammation analysis
To investigate the regulation of inflammation within the prostate, we conducted an analysis of mast cell recruitment to sites of inflammation.Notably, the presence of mast cells within the prostate was notably reduced in both the PC + LDE + DTX (p = 0.0394) and PC + LDE + DTX + Ex (p = 0.0139) groups when compared to the PC group (Fig. 5F, L).Furthermore, to establish a direct link between PC + LDE-DTX treatment and the modulation of the inflammatory state, we conducted immunohistochemistry staining and quantified the levels of key proteins involved in these processes.The animals treated with PC + LDE-DTX (p = 0.0010) and PC + LDE + DTX + Ex (p = 0.00122) exhibited markedly lower protein expression levels of IL-6 in stark contrast to the PC group (Fig. 5B,H).In relation the TNF-α, the PC + LDE + DTX group was reduced compared to PC + LDE + DTX (p = 0.0131), and PC + Ex (p = 0.0131; Fig. 5C,I).The PC group increased TNF-α in comparation to PC + LDE + DTX (p = 0.0453), and PC + LDE + DTX + Ex (p = 0.0001; Fig. 5C,I).NF-κB markers, in stark contrast to the PC in related to PC + LDE + DTX (p = 0.0109), and PC + LDE + DTX + Ex groups (p = 0.0083; Fig. 5D,J).
To ascertain the anti-inflammatory potential of the treatment on the prostate, we assessed interleukin-10 (IL-10) expression.IL-10, a cytokine crucial for maintaining immune response equilibrium, demonstrated enhanced expression levels in the prostate tissues of the PC + LDE-DTX (p = 0.0031), PC + LDE + DTX + Ex (p = 0.0169), and PC + Ex (p = 0.0050) groups in comparison to the PC group (Fig. 5E,K).

Discussion
To the best of our knowledge, this study represents the pioneering documentation of in vivo effects of a docetaxelloaded nanoparticle in prostate tumors, in association with physical exercise.Within this study, the administration of LDE-DTX demonstrated remarkable success in diminishing tumor volume in this rat-induced model of prostate cancer, all without discernible toxic effects.Rats afflicted with prostate cancer exhibited a substantial elevation in the incidence of invasive growth metastatic adenocarcinomas within the prostate 17 .Presumably, this model mirrors the aggressive tumor progression accompanied by escalated proliferation.Our findings are in concurrence with prior reports showcasing the impact on tumor volume attributed to LDE nanoparticle conjugation with chemotherapy agents 5,18 .We provide compelling evidence of LDE-docetaxel efficacy in reducing prostate mass, adenocarcinoma, and high-grade neoplasms.Another key finding of this study was the fact that aerobic physical exercise simultaneously to LDE-DTX treatment had the capacity of increasing the reduction of the tumor volume produced by LDE-DTX.Furthermore, in conjunction with physical exercise, LDE-docetaxel demonstrated an augmented internalization of LDE [14C]-cholesteryl ester within the prostate, contributing to further tumor volume reduction.The outcomes achieved through the potent antiproliferative agent DTX, in conjunction with LDE, hold the potential to illuminate novel pathways in the pursuit of overcoming prostate cancer, while ensuring treatment efficacy over extended periods.
As shown by our results, elevated levels of circulating cholesterol and lipids were positively linked to the development of PC.LDE-docetaxel treatment indicated a potential reduction in lipid levels in the blood regardless of whether they underwent physical exercise.Cancer cells show metabolic reprogramming, mainly affecting energy metabolism 19 .Nevertheless, neoplastic cells in prostate exhibit alterations in the metabolic pathways involving fatty acids, triglycerides, and cholesterol 20 .Furthermore, LDE-DTX treatment demonstrates elevated levels of HDL and decreased values of triglycerides and glucose.Data from a recent meta-analysis utilizing epidemiological studies found that high blood concentrations of cholesterol and triglycerides, coupled with low concentrations of HDL-C, are associated with an increased risk of overall cancer 21 .Experimental studies have shown that HDL-C can protect against tumor development through various mechanisms, such as influencing signaling pathways by modulating cholesterol content in cell membranes 22 .Experimental studies have also revealed that HDL-C possesses antioxidant and anti-inflammatory properties and plays a role in inhibiting the LDL oxidation cascade 23 .Thus, the protective effects of LDE-docetaxel through HDL may mitigate prostate cancer progression.Similarly, we have observed the metabolic effect of physical exercise, resulting in increased energy metabolism due to heightened muscular activity.Therefore, low levels of blood lipids could serve as an indirect measure of response to LDE-docetaxel treatment combined with exercise, reflecting a decrease in this metabolic activity with slowed tumor growth.These findings potentially hold translational significance, as they indicate that reducing cholesterol may be beneficial in lowering the risk of prostate cancer associated with LDE-DTX treatment.
We found a significant increase in the values related to the 646 cm −1 band and the 1003 cm −1 band by Raman spectroscopy related to tyrosine and phenylalanine in the groups treated with LDE-docetaxel with or without physical exercise.Metabolic biomarkers can be used for PC diagnosis.These compounds include amino acids such as phenylalanine and tyrosine, that were identified as diagnostic indicators for PC in body fluids 24,25 .Phenylalanine is the main metabolic pathway that yields the amino acid tyrosine, which is involved in the production of melanin and thyroxine.Tyrosine as a cancer marker has been studied in different types of cancer.Miyagi et al. 26 showed a significant decrease in tyrosine levels in plasma samples from patients with gastric cancer compared to controls, and the same pattern was also apparent between early and advanced stage gastric cancer.In the same way, Zhang et al. 27 showed decreased serum tyrosine levels in patients with esophageal cancer compared to healthy controls.Thus, it is possible to verify that the treatment with LDE-docetaxel increased the concentration of biomarkers indicating alteration in the metabolism of phenylalanine related to the reduction of the PC.Furthermore, we investigated the effect between LDE-docetaxel and physical exercise in increasing the activation of p53 pathways, which has been shown to play a central role in inducing apoptosis and interrupting DNA replication, thus mediating cell cycle arrest.Previous studies have indicated the crosstalk between p53 and AR signaling, where the overexpression of p53 inhibits expression of androgen-dependent genes 28 .Activation of p53 and subsequent transcription of several p53 target genes, including p21, induces long-term cell cycle arrest or apoptosis.Treatment with LDE-docetaxel associated with exercise considerably increased p53 expression and reduced AR levels in PC.After activation of p53 pathways, pro-apoptotic proteins (BAX and caspase 3) were up-regulated and anti-apoptotic proteins (BCL-2) were down-regulated in groups treated with LDE-DTX and LDE-DTX + Ex.Caspase expression and activation represent an important cellular marker of apoptosis 29 since the loss of apoptotic control in association with cell proliferation is responsible for the onset and progression of PC 30 .Altered caspase expression may represent an additional component related to cell death promoted by LDE-docetaxel treatment.Taken together, the data presented in this work indicates that LDE-docetaxel associated or not with physical exercise induces caspase-3-mediated apoptosis with changes in the BCL-2/BAX ratio.Therefore, our findings provide a link between LDE-docetaxel treatment and histopathological reduction of prostatic adenocarcinoma.By restricting these proteins, the proliferation, angiogenesis and anti-apoptosis abilities of neoplastic cells were all reduced.
For better understand the combined effect of LDE-docetaxel in an induced PC model, we analyzed cell cycle proteins after treatment.First, we observed a reduction in cell proliferation (Ki-67 expression) after treatment with LDE-docetaxel, reduced when associated with physical exercise (Fig. 3).To understand the effect of treatment on the cell cycle we analyzed cyclin D1 and CDK inhibitors p21 and p27, and we verified an increase in p21 and p27 protein in the treated groups.The p21 is a potent cyclin-dependent inhibitor that regulates G1/S phase progression and is tightly regulated by the tumor suppressor p53 31 .CDK 4 and CDK 6 are cyclin D1-regulated cell cycle proteins that are critical for cell cycle progression through G1 phase 32 .In our study we found the negative regulation of cyclin D1 and the positive regulation in the expression of p21 and p27 indicating a clear arrest of the G0/G1 cell cycle in the groups with LDE-docetaxel treatment and physical exercise.Considering that p21 and p27proteins inhibit mainly G1-S cyclin/Cdk complexes, we speculate that LDE-docetaxel may be affecting the cell cycle of PC, with modulation in proliferation arrest through disruption of the G1/S and G2/M transitions.However, the enhanced effect observed when the treatment with LDE-DTX was combined with aerobic physical exercise resulted in a significantly higher inhibition of cell cycle progression when analyzing the p53 protein, compared to treatment with LDE-docetaxel alone, which is innovative (Fig. 6).
In this study, LDE-DTX increased Nos2 and antioxidant enzymes.Within the tumor microenvironment, sustained supraphysiological concentrations of ROS overwhelm coping mechanisms, leading to a state of oxidative stress 33 .ROS generation is inherently countered by increased antioxidant capacity, where LDE + DTX nanoemulsion significantly elevated mRNA levels of Cat, Sod1, and Gss.The protocol used to induce tumors using DMBA is based on the premise of increasing harmful biological events, promoting changes in ROS 34 .ROS generated during treatment with LDE + DTX act as important subcellular messengers, stimulating various kinases involved in gene expression and cellular adaptation 35 .
Indeed, it is noteworthy that the action of LDE-DTX exhibited favorable outcomes across all molecular aspects of anti-carcinogenesis examined in this study.The expression of TNF-α and IL-6 proteins ranks among the most well-established pro-inflammatory cytokines, which were inhibited through LDE-DTX treatment in PC.Inflammation is closely intertwined with the development and progression of prostate cancer 36 .Treatment with LDE-DTX resulted in diminished mast cell quantities, coupled with reduced levels of IL-6, TNF-α, and NF-κB in PC.In this study, LDE-DTX treatment is associated with reduced adenocarcinoma, accompanied by decreased proliferation and increased apoptosis, as well as diminished inflammation, particularly when coupled with physical exercise.On the other hand, we discovered the anti-inflammatory effect of LDE-DTX, characterized by elevated levels of IL-10 in the prostate.IL-10 is recognized for its regulation of the immune system, suppressing exacerbation of the adaptive immune system response, and preventing tissue damage through immune response 37 .Administration of IL-10 to lymphoma-bearing mice significantly curtailed tumor growth, with a noteworthy rise in memory CD8 + T cells in comparison to non-IL-10-injected mice, suggesting IL-10's to enhance anti-tumor effects, immunity, and conceivably play a role in cancer recurrence prevention 38 .Hence, LDE-DTX and LDE-DTX + EX exhibit an immunosuppressive effect within the tumor microenvironment, modulated by both inflammatory and anti-inflammatory cytokines.
This article presents compelling evidence regarding the application of a chemotherapeutic drug-carrying nanoparticle, LDE, which exhibits a robust affinity for cellular LDL receptors.Our findings unequivocally demonstrate that LDE uptake is significantly augmented following physical exercise, subsequently exerting a profound influence on the metabolism of neoplastic cells (refer to Fig. 6).This research carries substantial significance in delineating an optimal treatment strategy for prostate cancer, as the synergistic interplay between physical exercise and small lipid particles, such as LDE with docetaxel, holds the potential to induce cell apoptosis through the p53 protein pathway in relation the PC and PC + Ex groups.Furthermore, the regulation of lipids and glucose utilization induced by physical exercise at a systemic level led to notable alterations in amino acid expression, thereby underscoring the extensive metabolic implications of the LDE-DTX and physical exercise interaction in PC development.The data elucidated in this study not only emphasizes the promising potential of LDE-DTX in prostate cancer treatment but also underscore its efficacy, but our findings unveil substantial reductions in tumor volume, diminished proliferative capacity, cell cycle arrest, and mitigation of inflammation.These outcomes underscore the significant therapeutic potential of LDE-DTX, hinting at its efficacy in restraining the progression of prostate cancer.Furthermore, the incorporation of physical exercise into the treatment regimen has the potential to amplify these effects, thereby unveiling avenues for complementary strategies that could further enhance patient outcomes.However, in conjunction with physical exercise and as a standalone therapeutic approach.

Conclusion
In conclusion the results of this study support the safety and effectiveness of LDE-DTX treatment to achieve prostate cancer regression in an experimental model of the disease.Increase in the anti-cancer action of LDE-DTX by simultaneous aerobic physical exercise was also shown.Those findings pave the way for future clinical studies aiming to introduce LDE-DTX in the oncological practice, with exercise training as an advisable supplementary measure to this drug-targeting therapy.

Animals and treatments
Sixty male Sprague-Dawley rats (40-day-old, weighing ± 120 g), were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at the University of Campinas (UNICAMP).Animals were housed 3-4 in polypropylene cages with laboratory-grade pine shavings as bedding and kept in a climatized room under controlled temperature at 22 ± 3 °C with a 12/12 h light/dark cycle, lights switched on at 7:00 a.m.During the procedures, standard rodent food (Nuvilab, Colombo, Paraná, Brazil) and filtered tap water were provided ad libitum.At 50 days of age, the animals were randomized in 6 groups and received a single dose of the carcinogen DMBA (Sigma Aldrich San Luis, Missouri, EUA®) intraperitoneal injection (i.p) of 65 mg/kg of body weight dissolved in sesame oil).During the period of tumor development, ultrasound was performed in the region to verify the development of the tumor mass (Supp 1).After the PC was developed (140-days-old) the animals designated to receive were administered i.p. 6 doses of LDE-docetaxel at 2 mg/kg/week.The animals assigned to perform aerobic physical training for 8 weeks started the treatment with LDE-docetaxel after the third week of the beginning of the physical training protocol.LDE-docetaxel dosages for the group submitted to physical exercise were performed 12 h after the last physical training session.The treatments occurred simultaneously (Fig. 1P).The animals were allocated top 6 experimental groups (n = 10): PC: rats with prostate cancer; PC + LDE*: rats with PC received single LDE dose; PC + LDE + Ex*: animals with PC received LDE single dose and exposed to aerobic physical training; PC + Ex: animals with PC was performed aerobic physical training for 8 weeks;

Incremental load test
The trained groups were submitted to the incremental load test to identify the maximum power of the animals, with this individualized data we stipulated an overload of 60% of the maximum speed, considered of moderate intensity for the performance of the aerobic physical exercise.The test was carried out at 141 days of age in the adaptation period of the animals on the treadmill (beginning), the second test after the 4th week of physical training (during), and the third test in the 8th week of physical exercise (final).The animals were adapted to aerobic physical exercise on a treadmill (Inbramed®, Presidente Prudente, São Paulo, Brazil).The incremental load test started with a speed of 6 m/min, with 0% inclination and increments of 3 m/min every 3 min performed until voluntary exhaustion of the rats.which occurred when the rats touched 5 times at the end of the treadmill in the period of 1 min.The maximum power (Pmax), defined as the animal's exhaustion speed (m/min), was used to prescribe the physical training intensities 39 .

Treadmill protocol
At 141 days of age, the rats started the adaptation protocol to aerobic physical exercise, and, at 147 days of age, the animals started aerobic physical training.The chronic aerobic physical exercise protocol was based on Ferreira 39 , consisting of 8 weeks, being each experimental week consisted of 5 consecutive days of training and 2 days of rest, with an overload of 60% of maximum speed.To readjust the load, incremental tests were carried out every 4 weeks.

Nutritional data
To monitor the biometric data, we assessed changes in food intake and body composition in the rats.Weekly measurements of body weight were taken, calculating the weekly weight change (Δ = final weight − initial weight).To determine the rat's total energy consumption, we used the food intake value and the caloric value of the rodent feed (3 kcal/g).The total energy consumption (TEI) was calculated as the average food consumption per day (in grams) multiplied by 3 (kcal/day = average food consumption per day [g] × 3).Additionally, we calculated the feed efficiency (EF) as the mean body weight gain divided by the total mean TEI (g/kcal = mean body weight gain/total mean TEI) 40 .We determined the amount of body fat by the sum of epididymal, retroperitoneal and mesenteric fat, data expressed in grams.

Biochemistry analysis
Blood samples were collected from the rats, then centrifuged and stored for subsequent biochemical assays.We measured concentrations of glucose, total cholesterol, HDL-cholesterol, and triglycerides using an enzymaticcolorimetric method, specifically the Trinder method.All procedures were performed in accordance with the established protocols provided by Gold Analisa Diagnóstica®, located in Belo Horizonte-MG, Brazil.

LDE uptake
To assess how LDE can reduce prostate cancer, we used an assay with radioactively labeled LDE and verified whether physical exercise would increase the percentage of uptake of the molecule.To perform this analysis, we used two groups: PC + LDE* (PC submitted to doses of LDE); PC + LDE + Ex* (PC and submitted to aerobic physical training and LDE doses for uptake analysis); the animals in the PC and PC + Exercise groups received an intraperitoneal injection of LDE labeled with OC-14C and after 24 h the tissues were collected and prepared

Figure 1 .
Figure 1.(A) Initial body mass (g) (B) Animals body weight referring only to the 8 weeks of physical exercise period (g); (C) Final body mass (g); (D) Weight gain referring only to the 8 weeks of physical exercise period (g/day); (E) Total weight gain along the experimental protocol (g/day); (F) Energy intake referring only to the 8 weeks of physical exercise period (Kcal/day); (G) Total energy intake (Kcal/day); (H) Feed efficiency referring only to the 8 weeks of physical exercise period (g/kcal); (I) Total feed efficiency (g/ Kcal); (J) Water consumption intake referring only to the 8 weeks of physical exercise period (mL/day); (K) Total fat of animals (g); (L) Tumoral volume (cm 3 ); (M) final ventral prostate weight (g); (N) Incremental load test of 3 moments (in the beginning of experiment, at the middle of experiment, and in the final of experimental protocol); (O) Difference between PC-Ex and LDE-DTX + Ex in incremental load test in 3rd moment; (P) Experimental delimitation related to protocol during the 14 weeks.

Figure 6 .
Figure 6.Graphical abstract that illustrates the treatment with LDE-docetaxel led to a decrease in cell proliferation with an increase in cellular apoptosis.This effect was amplified when combined with aerobic physical exercise.An increase in the cell cycle inhibitory proteins p-21 and p-27 can be observed, indicating an interruption in the G1/S and G2/M cell cycle transitions, suggesting a blockade in cell cycle progression at the G0/G1 phases.Elevated p-53 due to the treatment alters the apoptosis pathway by increasing the expression of BAX and caspase 3 and decreasing BCL-2.This image was created using Bio render software, https:// www.biore nder.com/. )

Table 1 .
Tissue uptake of the 14C-CE by tissues in groups PC and PC-Exercise rats after injection of the LDE.Values are means ± SD of uptake of LDE cholesteryl ether in % of CPM total/g of tissue.

Table 2 .
Occurrence of histopathological disorders in experimental animals with PC.The data are presented as the percentage, the significance of p < 0.05 is indicated by p value.The results were expressed as absolute value and percentage of occurrences, and the significant differences adopted were p < 0.05.